Composition and method for controlled release injections

ABSTRACT

The present invention is a pharmaceutical composition and method for controlling the release of a drug or vaccine to a patient where a slow, controlled release of drug or antigen occurs over a considerable period of time after injection. The drug or vaccine is contained in sugar glass microspheres and then placed in an anhydrous liquid, preferably perfluorocarbon, so that the vaccine is protected against dissolution while remaining surrounded by anhydrous liquid. This simple non-toxic system, deliverable by current syringe or present or future needle-free systems, is inexpensive and reliable and aids in parenteral drug delivery or mass immunization campaigns by reducing the need for repeated injections.

FIELD OF THE INVENTION

This invention relates generally to methods for controlled release ofinjected drugs and more specifically to controlled release vaccinations,which extend the duration of action of injected drugs or the duration oftriggering of the immune response long after infection by slowlyreleasing the drug or vaccine into the circulatory system.

BACKGROUND OF THE INVENTION

Currently, vaccinations include the need for multiple injections overtime in order to generate a protective memory immune response. Becausethe immune system responds only modestly to initial contact withantigens, repeat injections are necessary. Immunity is an adaptive orlearned process in which each subsequent exposure to antigen elicits astronger antibody response. The response is stronger, not only in thequantity of antibody made but also in the average affinity (the strengthof attachment or binding of the antibody for the antigen.) Affinityincreases because only those B cells which possess high-affinityreceptors are selectively triggered to proliferation and survival in thelater stages of the immune response as the concentration of antigenfalls. Early after injection, of course, the concentration of antigen ishigh enough to trigger both high and low affinity receptors. Withrepeated antigen injections, a greater number of the specific antibodyforming B lymphocytes are produced by this enhanced proliferation andsurvival as “memory” cells and the quantity of antibody thereforeincreases.

Typically, a childhood vaccination protocol for diphtheria, tetanus andpertussis (DTP) requires a priming dose of vaccine at 2 months of age, afirst booster injection at 4 months of age, a second booster at 6 monthsof age another dose at 15-18 months, and a recommended final dose at 4-6years of age (Centers for Disease Control and Prevention, NationalImmunization Program.) Other vaccines require similar protocols. Thesevaccine injections cause pain and distress, especially in infants;therefore, child-care providers often fail to return with the childrenfor later injections. As a result, the immunization protocol iscompromised and children are not properly protected against disease. TheWorld Health Organization (WHO) identified this failure of compliance asa widespread occurrence resulting in jeopardising mass immunizationcampaigns. (Jodar L., Aguado T., Lloyd J. and Lambert P-H (1998)Revolutionizing Immunizations Gen. Eng. News 18 p. 6.)

In order to address this problem, considerable efforts have been made todevelop techniques which reduce the number of injections required. Oneapproach is controlled release vaccines, which extend the duration oftriggering the immune response long after each injection, by slowlyreleasing the vaccine into the circulation. Most of the work to dateaddresses the tetanus vaccine encapsulated in bio-erodible plasticmicro-spheres of poly lactide/glycoloide polymers [(Xing D. K. L.,McLellan K., Corbel M. J., and Sesardic D. (1996) Estimation ofantigenic tetanus toxoid extracted from biodegradable microspheres.Biologicals 24, 57-65.] The biodegradable plastics slowly solubilize inbody fluids thereby releasing vaccine gradually from the erodedhydrophobic particles after injection. However, the vaccines were foundto be unstable in the body, therefore, early results were disappointing,but newer formulations overcame these problems and tetanus vaccine nowworks reasonably well in this system. Other fragile vaccines, however,are not stable in plastic particles in the body at 37° C. It is for thisreason that no other controlled release vaccine is currently in use.

In the course of developing stable liquids for injection, [(U.S. patentapplication Ser. No. 09/271,204 Composition and method for stableinjectable liquids] stabilized formulations of tetanus vaccine insoluble, sugar glass microspheres suspended in anhydrous oils orperfluorocarbon liquids were studied. The stabilizing agent used was asoluble glass of the sugar alcohol mannitol which, upon contact withbody water, it was expected to dissolve immediately and release itsvaccine as a conventional priming dose. In pre-clinical testing, thestable liquid formulations were injected subcutaneously into groups of10 guinea pigs. Dried vaccine was tested soon after manufacture (FIG. 1)and after it had been tested for stability by 3 months of acceleratedaging at 37° C. (FIG. 2).

Aliquots of dry vaccine, stabilized in sugar glass, were dissolved inwater before injection and served as controls as well as fresh sourcevaccine as the standard biological control vaccine preparation. Antibodyresponses were measured at 4, 8, and 12 weeks after injection.

It was found that all the control vaccines produced the typical kineticresponse of antibody titer after a priming dose, specifically, theantibody levels peaked at 4-8 weeks and then fell by 12 weeks. However,when both groups were injected with stabilized vaccine in glassmicrospheres suspended in anhydrous biocompatible liquids, the antibodylevels in these animals continued to rise throughout the whole of the 12weeks after injection. This result, not seen before in guinea pigtetanus toxoid immunization indicated a distinct change in the kineticsof antibody production. When compared with the historical results ofantibody kinetics studies in guinea pigs infected with repeated doses ofstandard vaccine, the present studies indicated controlled release ofantigen. The rising antibody levels seen in the present study at 12weeks indicated protection of the animals equivalent to that seen aftera multiple infection course of standard soluble vaccine.

While sugar glasses have proven to be efficacious in providing a stablemilieu for fragile biological molecules, other water-soluble glassessuch as the metal carboxylates and similar glasses (Slow ReleaseVitreous Systems PCT No. WO 90/11756) and the phosphate glasses(Phosphate Glass Ceramics for Biological na Medical Applications U.S.Pat. No 4,698,318) can also be formulated as glass microspheres, eitherin combination with sugar glasses or separately (Amorphous glasses forstabilizing sensitive products. PCT application WO 99/47174) and haveother desirable properties which suit them for use in this system. Thisincludes the ability to pre-determine the rate at which the glassdissolves by formulating it from a mixture of salts with individuallydifferent solubility rates in water (Controlled Delivery Devices U.S.Pat. No. 5,270,048).

The stable liquid formulations in anhydrous biocompatible liquids wereinitially developed to solve two major problems, namely the need torefrigerate drugs or vaccines for storage and the need to reconstitutethem in the field with sterile water before injection. Not only does thetechnology clearly overcome these drawbacks, it appears that theseformulations in anhydrous liquids also solve the compliance problem. Aconvenient/ready-to-inject, stable vaccine provides a complete course ofimmunization with a single injection in the present invention.

It is hypothesized that some of the soluble glass microspherescontaining the stabilized vaccine are protected against dissolution inbody water by remaining surrounded by anhydrous liquid. They dissolveonly at some time after injection to release their vaccine, which thenacts as a booster dose giving rising levels of antibody throughout thewhole 12 weeks of the experiment.

A lower average antibody level at 4 weeks exists with anhydrous liquidpreparations than with soluble antigen. This lower early titer in thegroups given anhydrous liquid suspensions of vaccine is arguably theresult of a lower dose of antigen being released soon after injection.Some may argue this indicates a problem of delayed onset of immunity,however the protective level of antibody in this system is approximately0.1 international units per milliliter of blood. The levels seen in theguinea pigs at 4 weeks were more than 1 international unit permilliliter, well above the protective level even at such an early stage.

BRIEF SUMMARY OF THE INVENTION

The present invention is a composition and method for controlled-releaseinjections using soluble glass microspheres suspended in anhydrousliquids such as oils, silicone fluids or perfluorocarbons where a slow,controlled dissolution of the microspheres and release of antigen occursover a considerable period of time after injection.

For a better understanding o f the present invention, together withother and further objects thereof, reference is made to the followingdescription, taken in conjunction with the accompanying drawings, andits scope will be pointed out in the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view of mean antibody titers of groups of 10 Guinea pigsinjected on day 0 with 0.5 ml vaccine and bled at 4, 8 and 12 weekslater.

FIG. 2 is a representation of mean antibody titers of groups of 10Guinea pigs injected on day 0 with 0.5 ml vaccine and bled at 4, 8 and12 weeks later.

DETAILED DESCRIPTION OF THE INVENTION

In the embodiment labeled FIG. 1, groups of 10 Guinea pigs were injectedon day 0 with 0.5 ml vaccine and bled at 4, 8 and 12 weeks later. In thefirst panel, animals were injected with fresh liquid vaccine from themanufacturer (Medeva batch T022). The mean antibody titre in animalsgiven fresh vaccine was higher than in the groups given dried vaccine,which suggests some loss of immunogenicity due to the drying protocol.Another 10 animals were injected with vaccine dried into a powder ofsugar glass microspheres and rehydrated with water immediately beforeinjection as indicated in the second panel. The third panel illustratesanimals injected with vaccine dried into a powder of sugar glassmicrospheres suspended in 0.5 ml squalane oil. The fourth panelillustrates animals injected with vaccine dried into a powder of sugarglass microspheres suspended in 0.5 ml perfluorodecalin. Finally, in thefifth panel, animals were injected with a powder of sugar glassmicrospheres not containing vaccine suspended in 0.5 ml of squalane andin the sixth panel animals were injected with a powder of sugar glassmicrospheres not containing vaccine suspended in 0.5 ml ofperfluorodecalin. While the antibody titre in the first two groups ofanimals injected with aqueous vaccines fell at 12 weeks after injection,the titre in the groups injected with vaccine in oil or PFC did not fallat 12 weeks. There was no antibody response in the fifth and sixthgroups of animals injected with the vehicle formulation only.

FIG. 2 illustrates an embodiment where mean antibody titers of groups of10 Guinea pigs were measured after they had been injected on day 0 with0.5 ml vaccine and bled at 4, 8 and 12 weeks later. In the first panel,animals were injected with fresh liquid vaccine from the manufacturerwhich had been stored at 40° C. for 3 months. Another group of animalswere injected with fresh liquid vaccine from the manufacturer which hadbeen stored at 37° C. for 3 months, as indicated in the second panel.The third panel represents animals injected with vaccine dried into apowder of sugar glass microspheres, stored at 37° C. for 3 months andrehydrated with water immediately before injection. The fourth panelillustrates animals injected with vaccine dried into a powder of sugarglass microspheres and suspended in 0.5 ml squalane and stored insqualane at 37° C. for 3 months before injection. Finally, the fifthpanel illustrates animals injected with vaccine dried into a powder ofsugar glass microspheres and suspended in 0.5 ml perfluorodecalin andstored in PFC at 37° C. for 3 months before injection. In thisparticular embodiment, the antibody titer in animals given fresh vaccinewas higher than in the groups given dried vaccine showing some loss ofimmunogenicity due to the drying protocol. While the antibody titer inthe two groups of animals injected with aqueous vaccines which had beenstored either wet or dry at 37° C. fell at 8 and 12 weeks afterinjection, the titer in the two groups injected with vaccine in oil orPFC rose progressively throughout the 12 weeks.

Experiments in vitro had suggested that PFCs might provide immediaterelease from glass microspheres suspended in them. They involvedincorporating a water-soluble dye (Mordant Blue 9) in the sugar glassmicrospheres, and suspending them at 10% w/v in perfluorodecalin. When0.5 ml of this opaque dark blue suspension was added to 1.5 ml of water,the PFC suspension fell to the bottom of the tube with the clear waterlayer on top. After vigorous vortex mixing for approximately one minuteand standing for an additional minute, the overlaying water layer becameintensely clear blue and the PFC layer cleared to water-white. Theclearing of the opacity showed that the suspended particles in the PFCliquid had dissolved. The migration of the blue coloration from the PFCto the water layer showed that all of the hydrophilic dye had dissolvedin the water as expected.

EXAMPLE 1

Using an easily assayed protein as a model vaccine confirms the aboveresults. The enzyme alkaline phosphatase was chosen. The followingsolution was made:

Adjuvant grade calcium phosphate 10% w/v (Superphos Kemi a/s);Trehalose, 10% w/v; ZnCl₂, 1 mM; MgCl₂, 1 mM; Alkaline phosphatase, 20U/ml in 5 mM Tris HCl buffer, pH 7.6.

The suspension was well mixed for 10 minutes at 37° C. to allow theinsoluble calcium phosphate to adsorb the soluble alkaline phosphatase.This absorption was measured by centrifuging small aliquots of thesuspension to deposit the calcium phosphate out of the suspension,sampling the supernatant solution and measuring its enzyme kineticsusing p-nitrophenyl phosphate as substrate and a wavelength of 405 nm.The remaining suspension was then spray-dried to produce a fine powder.Any desorption of the enzyme after rehydration of the powder wasmeasured in the supernatant as above. The powder was suspended at 20%w/v in perfluorophenanthrene and found to produce a stable suspension.

TABLE 1 Absorbance/min Sample Tested (405 nm) % Orginal solution (25 ul)0.409 100 Supernatant (25 ul) from above 0.034 8 Rehydrated powder 0.425104 (25 ul of a 20% w/v in water) Supernatant from above (25 ul) 0.004 120% w/v powder in PFC (25 ul) 0.430 105

Thus 92% of the enzyme was adsorbed to the calcium phosphate adjuvant(Table 1). All of this enzyme was eventually recovered for an assay ofenzyme activity after being suspended in PFCs in trehalose glassmicrospheres. These were re-dissolved in water as in the blue dyeexample above.

This experiment suggested that glass microspheres suspended in PFCsdissolved rapidly when mixed with water in vitro, indicating that thesepreparations would also release their antigen rapidly in vivo. This isapparently not the case. Surprisingly, there seems to be a slow,controlled release of antigen over a considerable period of time afterinjection. The release of antigen in this system is similar to thatthought to occur with certain oil-based adjuvant liquid emulsions usedin animals, such as Freund's Complete Adjuvant. It is thought that theslow leaking of the antigen from the droplets of antigen solutiondispersed in the mineral oil deposit of Freund 's adjuvant isresponsible for the greatly augmented immune responses found in animalsimmunized in this way. [(Freund J. Some aspects of active immunization.Ann. Rev. Microbiol 1 291 (1947).] Similar results have been found afterimmunization with antigens stabilized in sugar glass microspheressuspended in anhydrous biocompatible liquids. A major difference betweenFreund's adjuvant and the present system is that the former is a liquidemulsion of aqueous antigen solution droplets in oil and thereforeinherently unstable while the latter is a stabilized dry solid in aglass microsphere suspension and therefore inherently stable. Inaddition, Freund's adjuvant is a violent irritant and unacceptable foruse in humans [(Immunological adjuvants report of a WHO scientific groupmeeting held in Geneva from Oct. 6 to 10, 1975) 1976] while the PFCformulation used herein is non-toxic. It causes neither immediate nordelayed irritation or inflammation after injection. It is thereforeideally suited to the development of a single-dose vaccine for use inhumans, especially in children, where its lack of irritation is anadditional bonus. The ability of these formulations to control therelease of actives stabilized in soluble glass microspheres is not ofcourse restricted to vaccines. A wide variety of other drugs requirerepeated injections for their therapeutic efficacy. Indeed it isexceptional for a parenteral drug to be effective in a single dose. Ineach case the rate of release of the active molecule from solid solutionin the soluble glass into free solution in the body fluids would need tobe accurately controlled and would be different for each differentactive molecule.

While various anhydrous biocompatible liquids can be used in thissystem, PFCs are preferred because of their great chemical and physicalstability, their lack of toxicity, their low viscosity and surfacetension and their high density.

TABLE 2 Physicochemical properties of some perfluorocarbons. SurfaceVapor Density Viscosity Tension Pressure PFC MW (Kg/L) (mPas) (mN/m)(mbar) hexane 338 1.68 0.66 11.10 294 n-octane 438 1.73 .127 16.98 52decalin 462 1.92 5.10 17.60 8.8 phenanthrene 624 2.03 28.40 19.00 <1

A wide variety of PFC liquids can be obtained depending on theparticular parent hydrocarbon molecule that is fluorinated. It is likelythat the rate of absorption from the tissues into the bloodstream, andof removal from the body in the exhaled breath is a function of thevapor pressure of the PFC at body temperature. This is in turn generallyproportional to molecular weight (Table 2). By carefully choosing aparticular PFC, it is likely that the rate of controlled release can bevaried over a substantial range. Since the PFC liquids can also beblended together, the release rates can be precisely fixed by choosingan appropriate mixture of PFC liquids.

Calcium phosphate, used as a density matching agent, is insoluble inwater and forms a fine, highly-hydrated colloidal suspension which isable to reversibly bind large amounts of macromolecules, especiallyproteins, from solution. A significant proportion of the antigen presentin the tetanus toxoid vaccine used in these studies was bound to boththe aluminum hydroxide adjuvant originally used in the vaccine and tothe calcium phosphate suspension used as a density matching substance.The protein antigens bound to these inorganic colloids act as areservoir for the sustained release seen in the animal studies. Thedegree of delay in the release profile seems to be much greater when thevaccine is in suspension in PFC liquids than with the aqueoussuspension, used as a control. This suggests that the non-aqueous PFCliquid is the critical component in controlling the rate of release fromthe inorganic colloids. It will require further experimentation toestablish whether equally sustained release from PFC liquids can occurif the protein is free in solid solution in the glass microspheresrather than bound to an inorganic colloid.

The use of calcium phosphate in these formulations has additionaladvantages over and above the matching of the density of the sugar glassmicrospheres with the PFC liquids. Since the inorganic fraction of boneitself consists of calcium phosphate in the form of hydroxyapatite, thechemistry of this additive is biocompatible and non-toxic. It is safe toassume that calcium phosphate injected in this way will be locallynon-toxic and will be slowly solubilized from the injection site and/orthe regional draining lymph nodes eventually, leaving no excess.

Before this occurs, the deposit of calcium phosphate at the injectionsite acts as a positive marker of immunization, which is detectable bymedical imaging techniques, such as x-rays or MRI, or even possibly byultrasound or magnetometry. The ability to positively identify patientswho have been immunized is sometimes of real importance in diseaseeradication programs where local record keeping is imperfect andpatients' knowledge of their own immunization history may be incomplete.By substituting other density-regulating materials such as bariumsulphate, titanium dioxide and other insoluble and dense inorganicprecipitates or defined mixtures of them, it may be possible to uniquelymark different vaccines with separate density matching chemicals. Thenan accurate immunization history may be detectable by relativelysuperficial medical imaging or detection methods.

While there has been described what are believed to be the preferredembodiments of the present invention, those skilled in the art willrecognize that other and further changes and modifications may be madethereto without departing from the spirit of the invention, and it isintended to claim all such changes and modifications as fall within thetrue scope of the invention.

What is claimed is:
 1. A pharmaceutical composition comprising a drugselected from the group consisting of hormones, analgesics, narcotics,narcotic antagonists, chemotherapeutics, immunosuppressants,immunomodulators, contraceptives, vasoactive agents, coagulationmodifiers, cardioactives, anti-inflammatories, and CNS drugs to beinjected into a patient wherein, the drug is in soluble glass compositemicrospheres of sugar and calcium phosphate and the soluble glasscomposite microspheres are suspended in a biocompatible anhydrous liquidwhereby the drug is protected against dissolution while remainingsurrounded by anhydrous liquid thereby, extending the duration of actionof the drug long after injection by slowly releasing the drug or vaccineinto the patient's circulatory system.
 2. The pharmaceutical compositionaccording to claim 1 wherein said vaccine is selected from a groupconsisting of toxins, toxoids, live or killed bacteria, live or killedviruses, live or killed protozoa, recombinant proteins, DNA, RNA,polysaccharides, lipoproteins and lipids and recombinant or syntheticpeptides.
 3. The pharmaceutical composition according to claim 1 whereinsaid soluble glass microspheres are selected from a group consisting ofnon-reducing sugars and sugar alcohols, metal carboxylates and phosphateglasses.
 4. The pharmaceutical composition according to claim 3 whereinsaid non-reducing sugars are selected from the group consisting ofsucrose, trehalose, raffinose, and stachyose.
 5. The pharmaceuticalcomposition according to claim 3 wherein said sugar alcohols areselected from the group consisting of mannitol, arabinitol, inositol,glucitol, galactitol, xylitol, maltitol, lactitol, glucopyranosylsorbitol and glucopyranosyl mannitol.
 6. The pharmaceutical compositionaccording to claim 1 wherein said anhydrous biocompatible liquid isselected from a group consisting of anhydrous hydrophilic liquids,anhydrous hydrophobic liquids, anhydrous silicone fluids or anhydrousperfluorocarbons.
 7. The pharmaceutical composition according to claim 1wherein said glass composite microspheres contain an amount of aninsoluble biocompatible high-density agent sufficient to raise theaverage density of the microspheres to match that of the anhydrousbiocompatible liquid in which they are suspended.
 8. The pharmaceuticalcomposition according to claim 7 wherein said insoluble biocompatiblehigh-density agent is selected from the group consisting of calciumphosphate, aluminum phosphate, aluminum hydroxide, barium sulphate andtitanium dioxide.
 9. The pharmaceutical composition according to claim1, wherein said vaccine is tetanus toxoid.
 10. The pharmaceuticalcomposition according to claim 9, wherein said vaccine is adsorbed to anadjuvant.
 11. The pharmaceutical composition according to claim 10,wherein said adjuvant is selected from a group consisting of aluminumhydroxide, aluminum phosphate or calcium phosphate.
 12. Thepharmaceutical composition according to claim 9, wherein a suspension ofcalcium phosphate colloidal gel is added to a solution of trehalose,zinc chloride, magnesium chloride, and Tris buffer which is added to aidin the formation of a stabilized tetanus toxoid in said glassmicrospheres.
 13. The pharmaceutical composition according to claim 12,wherein said solution is spray-dried to a fine-powder.
 14. Thepharmaceutical composition according to claim 13, wherein said powder issuspended in perfluorocarbon to produce a stable suspension.
 15. Thepharmaceutical composition according to claim 14, wherein saidperfluorocarbon is selected from a group consisting of perfluorohexane,perfluorodecalin, perfluorooctane and perfluorophenanthrene.
 16. Amethod of formulating a drug or vaccine to prolong the duration ofaction when administered in an effective amount, which drug or vaccineis formulated by: incorporating said drug or vaccine in soluble glasscomposite microspheres suspending said soluble glass compositemicrospheres in a biocompatible anhydrous liquid whereby said drug orvaccine is protected against dissolution while remaining surrounded byanhydrous liquid thereby slowly releasing the drug or vaccine into thepatient's circulatory system.
 17. The method of claim 16 including thestep of producing the soluble glass composite microspheres is selectedfrom the group consisting of spray-drying, air drying, vacuum drying,emulsion solidification, precipitation, and melting and grinding to afine powder.
 18. The method of claim 17 wherein said fine powder issuspended in the anhydrous biocompatible liquid which is aperfluorocarbon.
 19. The method of claim 18 wherein said perfluorocarbonis selected from the group consisting of perfluorohexane,perfluorodecalin, perfluorooctane and perfluorophenanthrene.